WO2004095033A2 - Compositions et procedes de diagnostic de grossesse precoce precise chez les ongules - Google Patents

Compositions et procedes de diagnostic de grossesse precoce precise chez les ongules Download PDF

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WO2004095033A2
WO2004095033A2 PCT/US2004/011456 US2004011456W WO2004095033A2 WO 2004095033 A2 WO2004095033 A2 WO 2004095033A2 US 2004011456 W US2004011456 W US 2004011456W WO 2004095033 A2 WO2004095033 A2 WO 2004095033A2
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ucrp
seq
amino acids
amino acid
antibody
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WO2004095033A3 (fr
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Nagappan Mathialagan
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Mosanto Technology Llc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Definitions

  • This invention relates generally to the fields of veterinary medicine, reproductive biology and diagnostics, and more specifically, improved methods for early stage pregnancy detection in ungulates (hoofed animals), including ruminants such as bovines, e.g., dairy and beef cattle, and ovines.
  • ruminants such as bovines, e.g., dairy and beef cattle, and ovines.
  • Pregnancy diagnosis is an important component of sound reproductive management, particularly in the dairy industry (Oltenacu et al, 1990), where a high proportion of artificial inseminations fail (Streenan and Diskin, 1986).
  • a reliable yet simple pregnancy test for cattle has long been sought.
  • Several procedures are available, including a milk progesterone assay (Oltenacu et al, 1990; Markusfeld etal, 1990), estrone sulfate analysis (Holdsworth et al., 1982; Warnick et al., 1995), rectal palpation (Hatzidakis et al., 1993), ultrasound (Beal et al., 1992; Cameron and Malmo, 1993), and blood tests for pregnancy-specific antigens.
  • progesterone milk assay is said to be the most cost-effective for the producer (Oltenacu et al., 1990; Markusfeld etal, 1990). Next best is rectal palpation performed at day 50 post- insemination (Oltenacu et al, 1990).
  • IFN-tau interferon-tau
  • the protein was found to be similar in size to human IFN-stimulated gene product- 15 (also called huUCRP) and to be immunoreactive with anti-serum against ubiquitin.
  • This protein has, therefore, been termed bovine ubiquitin cross-reactive protein ("UCRP") (Austin et al. 1996).
  • UCRP bovine ubiquitin cross-reactive protein
  • amino acid sequence accesion No. AAB57687
  • nucleotide sequence accesion No. U96014
  • the bovine UCRP amino acid sequence is listed herein as SEQ ID NO:34 with the corresponding nucleotide sequence listed for reference as SEQ ID NO:35. All UCRP numbering referred to herein is based upon the amino acid sequence numbering from SEQ ID NO:34 (Accession No. AAB57687, Austin et al. 1997).
  • Bovine UCRP also known as bovine IFN-stimulated gene product- 17, or ISG17
  • ISG17 bovine IFN-stimulated gene product- 17,
  • IFN-tau secreted by the trophoblast layer of the conceptus functions as a maternal signal denoting recognition of pregnancy in ruminants.
  • IFN-tau belongs to Type I IFN class of proteins (IFN-alpha and IFN-beta). IFN alpha and IFN beta also interact with Type I receptors on the cellular membranes and utilize similar signal transduction pathways. Receptors for IFN-tau are present on the endometrial epithelial cells of the uterus. Sequencing and binding studies have shown that these IFN-tau receptors belong to IFN Type I receptor class. [0009] Hansen et al, 1997 reports the pregnancy specific secretion of ISG17 by the endometrium during days 15 to 26 of pregnancy in response to conceptus-derived IFN-tau.
  • This invention provides methods for the early detection of pregnancy in livestock such as ungulates (e.g., hoofed animals).
  • methods are provided for the early detection of pregnancy in ruminants, e.g., a bovine or ovine animal suspected of being pregnant, comprising obtaining a sample from the animal, measuring the level of UCRP in the sample with an antibody prepared by any of the methods described herein, wherein elevated levels of UCRP indicate that the animal is pregnant.
  • the sample may be of any biological material including saliva, urine, or milk, and is preferably whole blood, serum, or plasma.
  • the sample may be obtained from the animal from about day 15 to 30 post-insemination, preferably from day 15 to 28, more desirably from day 15 to 25 or,from day 15 to about 20 post-insemination, including most advantageously from about day 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 post-insemination.
  • One aspect of the present invention includes methods for predicting antigenic sequences of an ubiquitin cross-reactive protein (UCRP) comprising: (a) selecting an analysis program that predicts antigenic amino acid sequences; (b) inputting the UCRP amino acid sequence into the analysis program; (c) obtaining the predicted antigenic amino acid sequences; (d) aligning the UCRP sequences with protein sequences for which X-ray crystal structure data is known and that share at least 80% sequence identity with said UCRP protein to obtain predicted X-ray crystal structure data for UCRP; (e) comparing the predicted antigenic amino acid sequences of the UCRP protein obtained in step (c) to the three dimensional X-ray crystal structure data of step (d); and (f) identifying antigenic UCRP amino acid sequences based upon the comparative data from step (e).
  • UCRP ubiquitin cross-reactive protein
  • step (d) it is contemplated that the UCRP protein and aligned protein sequences will share at least 80% sequence identity; however, it is also dersirable that the (d) proteins share even greater sequence identiy, including from 80-100% identity, preferably at least 85% identity, or more preferably at least 90% identity. It is also contemplated that the proteins aligned in step (d) share 90-95% identity, or even more preferably, that they share at least 95% identity. It is also contemplated that the shared identity be exhibited in regions or domains of the protein (contrasted to the entire protein sequence), ranging from at least three amino acids, and typically the domains or regions may be from three to twenty amino acid residues in length, or greater.
  • the method also includes identifying antigenic UCRP amino acid sequences that are within at least one of six antigenic regions comprising:
  • the method further includes identifying antigenic UCRP amino acid sequences that are selected from the group consisting of: SEQ ID NO:l, (GDLTV, amino acids 3-7 of UCRP); SEQ ID O:2, ( LGGQEI, amino acids 10-15 of UCRP); SEQ ID O:3, (LRDSM, amino acids 19-23 of UCRP); SEQ ID NO:4, (RDSMT, amino acids 20-24 of UCRP); SEQ ID NO:5, (KQF, amino acids 29-31 of UCRP); SEQ ID NO:6, (PAFQQR, amino acids 39-44 of UCRP); SEQ ID NO:7, (QGLRAG, amino acids 63-68 of UCRP); SEQ ID NO:8, (LVVQNC, amino acids 73-78 of UCRP); SEQ ID NO: 9, (ISILVR, amino acids 79-84 of UCRP); SEQ ID NO: 10, (RNDKGR, amino acids 84-89 of UCRP); SEQ ID NO: 11, (
  • Another aspect of the invention includes antibodies generated by using at least one of the antigenic UCRP amino acid sequences identified by the above method for predicting antigenic sequences of UCRP, as an immunogen. These antibodies include those that are generated using antigenic UCRP amino acid sequences that are within at least one of six antigenic regions comprising:
  • the antibodies that are useful in this invention include those that have been generated by using one or more of the following amino acid sequences as an immunogen, preferably the antigenic sequence is similar to epitopes or antigenic regions from within full-length ungulate/bovine UCRP, and preferably the UCRP antigenic sequences contain a surface portion of a three-dimensional UCRP protein conformation as it would normally be found in the animal including the following amino acid sequences: SEQ ID NO: 1, (GDLTV, amino acids 3-7 of UCRP);
  • SEQ ID NO: 2 (LGGQEI, amino acids 10-15 of UCRP);
  • SEQ ID NO: 7 (QGLRAG, amino acids 63-68 of UCRP);
  • SEQ ID NO: 10 (RNDKGR, amino acids 84-89 of UCRP);
  • SEQ ID NO: 15 (EEYG, amino acids 135-138 of UCRP);
  • SEQ ID NO:16 (YGLMK, amino acids 137-141 of UCRP);
  • SEQ ID NO: 17, (MKG, amino acids 140-142 of UCRP);
  • SEQ ID NO:18 (NLRLRG, amino acids 148-153 of UCRP);
  • SEQ ID NO:20 (DSRE, amino acids 49-52 of UCRP);
  • SEQ ID NO:21 (QTVA, amino acids 99-102 of UCRP);
  • SEQ ID NO:24 (MDDEH, amino acids 128-132 of UCRP);
  • amino acid sequences, residues, fragments, regions, and epitopes are used throughout the description to identify certain desired amino acid sequences that may exhibit any one or combination of desired characteristics including being antigenic, surface exposed, and exhibiting a useful conformation that is found in the native UCRP protein.
  • amino acid sequences, residues, and fragments are used synonymously.
  • Antibodies useful in this invention can be generated by using more than one of the above- listed amino acid sequences as an immunogen. Each of such antibodies can, and commonly does, bind to or immunoreacts with amino acid sequences within full-length UCRP that are longer than but contain one or more of the above-listed sequences.
  • antibodies useful in this invention may bind to or immunoreact with the following sequences: (a) SEQ ID NO:5 and an additional L (amino acid 28 of UCRP) or EL (amino acids 27-28 of UCRP) and/or an additional IA (amino acids 32-33 of UCRP); (b) SEQ ID NO: 15 and an additional L (amino acid 134 of UCRP) and/or an additional L (amino acid 139 of UCRP); (c) SEQ ID NO:17 and an additional C (amino acid 143 of UCRP); (d) SEQ ID NO: 19 and an additional R (amino acid 44 of UCRP); SEQ ID NO: 21 and an additional K (amino acid 98 of UCRP) or LK (amino acids 97-98 of UCRP) and/or an additional E (amino acid 103 of UCRP); SEQ ID NO:22 and an additional V (amino acid 108
  • Antibodies useful in this invention can be generated using more than one of the above- listed amino acid sequences as the immunogen.
  • antibodies useful in specific embodiments of the invention are generated by using at least two (in some cases preferably three) of the following combinations of such amino acid sequences as the immunogen for generating an antibody: SEQ ID NOs: 1, 3 and 7; SEQ ID NOs: 2, 6 and 8; SEQ ID NOs: 9, 13 and 16; and SEQ ID NOs: 12, 14 and 18.
  • Each of such antibodies can, and commonly do bind, alternatively or also, to amino acid sequences within full-length UCRP that are longer than but contain one or more of the above-listed sequences.
  • UCRP is present in early pregnancy and, at about two months (preferably about one month) post-partum, is essentially absent (undetectable by methods commonly available at this time) or at least not present above a "background” or “baseline” level that can be measured and established as a reference point against which the UCRP levels that are detected in accordance with this invention can be evaluated as being “elevated” or “not elevated.”
  • Antibodies useful in this invention can include monoclonal antibodies or polyclonal antisera.
  • the immunologic detection may be carried out using any suitable techniques, including ELISA, RIA, and Western blot.
  • the ELISA may comprise a sandwich ELISA comprising binding of UCRP to a first antibody preparation fixed to a substrate and a second antibody preparation labeled with an enzyme.
  • the enzyme is alkaline phosphatase or horseradish peroxidase.
  • an elevated level of UCRP is any amount greater than about 0.5 ng/ml of serum.
  • elevated levels range from about 0.5 ng/ml to about 10 ng/ml of serum, and more commonly from about 1 ng/ml to about 4 ng/ml.
  • any UCRP level that is above about 0.5 ng/ml is considered to be elevated above baseline, that is elevated above the level of UCRP in a non-pregnant ungulate animal.
  • the invention provides a method of making a breeding decision for a bovine animal comprising: using at least one of the antibodies of this invention to determine whether the level of UCRP in a sample from an animal suspected of being pregnant is elevated, wherein (i) elevated levels of UCRP indicate that the animal is pregnant, and no further steps need be taken; and (ii) non-elevated levels of UCRP indicate that the bovine animal is not pregnant, and should be treated to impregnate in a timely manner, e.g., injected with gonadotropin-releasing hormone (GnRH), and about seven days later, injected with prostaglandin F 2 ⁇ (PGF), followed by re-insemination.
  • the method may comprise, about 48 hours after the PGF injection and before re-insemination, administering a second injection of GnRH.
  • This invention overcomes limitations of the prior art by enabling the more convenient preparation of antibodies useful in methods for reliably determining early pregnancy.
  • An accurate yet simple pregnancy test for cattle has long been sought.
  • commercially available tests have either not allowed early detection of pregnancy or have suffered from a high incidence of false positive or false negative results.
  • the prior tests, although potentially useful, have fallen short of expectations in terms of their practical, on-farm value and use.
  • Antibodies useful in this invention can be prepared using any of various techniques well known in the art, and by using UCRP sequences or fragments that have three-dimensional protein conformations suitable for generating antibodies that faithfully react with full-length (native) UCRP as immunogens, as described herein.
  • such fragments can be synthesized more economically than purifying enough full-length UCRP to prepare the antibodies, or by synthesizing full-length UCRP, e.g., by the use of recombinant DNA, for that purpose. However, if desired, these antibodies can be raised against such full-length UCRP. I. Identification of antigenic fragments of UCRP.
  • bovine UCRP also referred to as ISG17
  • ISG17 The desired, antigenic sequences of bovine UCRP
  • the UCRP sequence analysis was performed using the ProteanTM component of the DNA StarTM sequence analysis package.
  • the ProteanTM program provides a series of software packages that determine the structural features of input protein sequences.
  • the algorithm used for the analysis predicts the secondary structural features such as alpha-helix and beta sheet, hydrophobicity and surface probability of amino acid residues.
  • Four different algorithms from the ProteanTM program were used to generate data for predicting antigenic sites of the UCRP protein, including the Sette MHC motifs (Sette et al. 1989), the AMPHI analysis (Margalit et al.
  • the Sette MHC motif analysis predicts peptide antigenic sites that interact with mouse MHC II haplotype d proteins.
  • the AMPHI analysis predicts immunodominant helper-T-lymphocyte antigenic sites from primary sequence data.
  • the Rothbard-Taylor analysis locates potential T-lymphocyte antigenic determinants that contain a common sequence motif.
  • the Jameson- Wolf analysis predicts potential antigenic determinants by combining existing methods for protein structural predictions including hydrophobicity Hopp-Woods values (Hopp and Woods, 1981), (Hopp- Woods, H [ ⁇ 2>0.5 ⁇ -l>-0.5 ⁇ -2>]), surface probability values of Emini (Emini et al. 1985), (Emini, S [ ⁇ 1>1.0 ⁇ 0>]), flexibility values of Karplus-Schultz (Karplus et al.
  • Region II SEQ ID NO:29, which consists of UCRP amino acid residues 19-33: LRDSMTVSELKQFIA (residues 19-29 were predicted by Jameson- Wolf; the overlapping sequence motifs of residues 28-33 and 27-31, were predicted by Sette and AMPHI, respectively); [0034] Region III.SEQ ID NO: 30, which consists of UCRP amino acid residues 44-55:
  • CDD conserved domain database
  • the CDD search with the UCRP sequence identified a number of proteins that shared conserved sequence domains with the UCRP sequence, and for which X-ray crystal structure data is available. These proteins included Sumo-1 (Accession No. 1A5R, GI 3892023); Nedd8 (Accession No. Q15843), and Smt3 (Accession No. Q12306). The X-ray crystal structures for these three proteins have been solved and are available on the NCBI web site. [0041] The conserved domain database search with the UCRP protein sequences identified six hits, with the best alignment to the conserved domain being "pfam00240.7,” ubiquitin, and PSSM-Id:7525.
  • UCRP amino acid residues 5-78 and 79-154 exhibited 97% and 100% alignment with the sequence of Smt3, suggesting that UCRP shares at least two domains with the Smt3 protein. Each of these apparently shared domains consists of about 75 amino acid residues and is referred to as a "conformational specific region or epitope”.
  • the conserved domain database analysis program can be used to effectively substitute the amino acid residues of a desired protein, in this case, UCRP, into the crystal coordinates of the known structure, such as Smt3 in the present example.
  • UCRP query protein
  • Smt3 conserved domain database hit
  • This CDD analysis program also enables one to view the results of such a prediction by providing an aligned view of the conserved domains using the C3nD program.
  • Conformation specific domain 1 SEQ ID NO:l (UCRP amino acids 3-7, GDLTV); SEQ ID NO:3 (UCRP amino acids 19-23, LRDSM); and SEQ ID NO: 7 (UCRP amino acids 63-68, QGLRAG); Conformation specific domain 2: SEQ ID NO: 2 (UCRP amino acids 10-15, LGGQEI); SEQ ID NO: 6, (UCRP amino acids 39-44, PAFQQR); and SEQ ID NO:8, (UCRP amino acids 73-78, LVVQNC); Conformation specific domain 3: SEQ ID NO: 9 (UCRP amino acids 79-84, ISILVR);
  • SEQ ID NO: 13 (UCRP amino acids 95-99, VQLKQ); SEQ ID NO: 16 (UCRP amino acids 137-141 (YGLMK); Conformation specific domain 4: SEQ ID NO: 12 (UCRP amino acids 87-91, KGRSS); SEQ ID NO: 14 (UCRP amino acids 115-120, QADQFW); and SEQ ID NO: 18, 148-153 (NLRLRG).
  • any amino acid sequence or fragment from within any region of the six predicted antigenic regions (Regions I-VI), or conformation specific domains one through four will be useful an immunogen or in any combination as immunogens for generating UCRP specific antibodies of the present invention.
  • each one of these combinations may represent specific conformational regions, surface exposed sequences, or epitopes of UCRP that would be good targets for the generation of specific UCRP reactive antibodies of the present invention.
  • the present invention entails the use of antibodies in the immunologic detection of UCRP.
  • Various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. 1987.
  • Immunoassays in their most simple and direct sense, are binding assays.
  • Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA).
  • immunochromatographic assays including those known in the art as a "lateral flow assay", which can be defined as an immunochromatographic determination of the presence or absence of an antigen in a sample from an animal by combining the sample with a coloring agent-coupled antibody to the antigen, allowing the resulting combination to migrate into a first region containing a second portion of the antibody which is not coupled to a coloring agent, so that the appearance of color in the first region indicates that the antigen is present in the sample, and allowing the combination to migrate from the first region into a second region containing an antibody to the first antibody, so that the appearance of color in the second region, together with the absence of color in the first region, indicates that the antigen is not present in the sample.
  • this invention is not limited to the use of such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used in connection with the present invention.
  • antibody-based methods for detecting an antigen include obtaining a sample suspected of containing a protein, peptide or antibody, and contacting the sample with an antibody or protein or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of antigen-antibody complexes.
  • Contacting the chosen biological sample with the protein, peptide or antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with UCRP.
  • the UCRP antibody mixture will be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • detection of antigen-antibody complexes is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art.
  • U.S. Patents concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference.
  • the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the UCRP-specific first antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the UCRP antibody is used to form secondary immune complexes, as described above.
  • the second binding ligand contains an enzyme capable of processing a substrate to a detectable product and, hence, amplifying signal over time. After washing, the secondary immune complexes are contacted with substrate, permitting detection.
  • ELISA enzyme-linked immunoassay
  • the sensitivity of ELISA methods is dependent on the turnover of the enzyme used and the ease of detection of the product of the enzyme reaction. Enhancement of the sensitivity of these assay systems can be achieved by the use of fluorescent and radioactive substrates for the enzymes.
  • Immunoassays encompassed by the present invention include, but are not limited to those described in U.S. Patent 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent 4,452,901 (western blot), both incorporated herein by reference. Other assays include immuno-precipitation of labeled ligands and immuno-cytochemistry, both in vitro and in vivo.
  • the invention comprises a "sandwich" ELISA, where anti- UCRP antibodies are immobilized onto a selected surface, such as a well in a polystyrene microtiter plate or a dipstick. Then, a test composition suspected of containing UCRP, e.g., a clinical sample, is contacted with the surface. After binding and washing to remove non- specifically bound proteins, the bound antigen may be detected by a secondary antibody to the UCRP.
  • a test composition suspected of containing UCRP e.g., a clinical sample
  • polypeptides from the sample are immobilized onto a surface and then contacted with the anti-UCRP antibodies. After binding and washing to remove non-specifically bound proteins, the bound antibody is detected. Where the initial antibodies are linked to a detectable label, the primary immune complexes may be detected directly. Alternatively, the immune complexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which the UCRP is immobilized involves the use of antibody competition in the detection. In this ELISA, labeled antibodies are added to the wells, allowed to bind to the UCRP, and detected by means of their label.
  • the amount of UCRP in a sample is determined by mixing the sample with the labeled antibodies before or during incubation with coated wells.
  • the presence of UCRP in the sample acts to reduce the amount of antibody available for binding to the well, and thus reduces the ultimate signal.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes.
  • coating a plate with either antigen or antibody one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder or rabbit serum.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • a secondary or tertiary detection means rather than a direct procedure.
  • the immobilizing surface is contacted with the control human cancer and/or clinical or biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG), evaporated or powdered milk, and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • solutions such as BSA, bovine gamma globulin (BGG), evaporated or powdered milk, and phosphate buffered saline (PBS)/Tween.
  • the "suitable" conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about lh to 2h to 4 h, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.
  • the second or third antibody will have an associated label to allow detection.
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for
  • the amount of label is quantified, e.g, by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H O 2 , in the case of peroxidase as the enzyme label.
  • Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • a variant of ELISA is the enzyme-linked coagulation assay, or ELCA (U.S. Patent

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Abstract

L'invention concerne des procédés améliorés de détection précoce de la grossesse chez les ongulés (p.ex., bovins) et de nouveaux anticorps utiles dans de tels dosages biologiques. Les anticorps sont générés à l'aide de séquences d'acides aminés spécifiques dans la protéine à réaction croisée vis-à-vis de l'ubiquitine (UCRP), en tant qu'immunogènes. Les niveaux élevés de protéines UCRP, lorsqu'ils sont détectés dans un échantillon (p.ex. les fluides organiques) de l'animal à partir des 15-30 et notamment des 15-28 jours après insémination, indiquent que l'animal est gravide. L'invention peut permettre d'augmenter efficacement les programmes de reproduction des animaux de ferme commerciaux.
PCT/US2004/011456 2003-04-17 2004-04-14 Compositions et procedes de diagnostic de grossesse precoce precise chez les ongules WO2004095033A2 (fr)

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Publication number Priority date Publication date Assignee Title
EP1546716A2 (fr) * 2002-05-02 2005-06-29 Aspenbio, Inc. Detection de la gravidite
EP1546716A4 (fr) * 2002-05-02 2007-04-11 Aspenbio Inc Detection de la gravidite
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